This invention relates to methods and compositions for the selective modification of nucleic acids in biological compositions.
Transmission of viral diseases (e.g., hepatitis A and B, acquired immunodeficiency syndrome (HIV), cytomegalovirus infections) by blood or blood products is a significant problem in medicine. While donor selection criteria and screening of donor blood for viral markers helps reduce the transmission of viruses to recipients, screening methods are incomplete or less than 100% sensitive, as most are directed to only a few discrete viruses. Even in such cases, their sensitivity is insufficient. In addition, other biological compositions, e.g., mammalian and hybridoma cell lines, products of cell lines, milk, colostrum and sperm, can contain infectious viruses.
It is desirable to inactivate any virus contained in donor blood, blood products, or other biological compositions. At the same time, it is important to leave the structure and function of valuable constituents, such as red blood cells, platelets, leukocytes, and plasma biopolymers, such as proteins and polysaccharides relatively unchanged.
In addition, it is often unknown whether compositions containing blood, blood products, or products of mammalian cells contain infectious viruses. In this case it would be valuable to have compositions and methods to treat such compositions to inactivate any infectious viruses present.
Furthermore, the manufacture of maximally safe and effective killed vaccines for human or veterinary use requires methods which completely and reliably render live microorganisms, e.g., viruses and bacteria, noninfectious (xe2x80x9cinactivatedxe2x80x9d) but which have minimal effects on their immunogenicity. Methods typically used for the inactivation of viruses, such as those useful in the preparation of viral vaccines, generally alter or destroy the function and structure of cells, cell components, proteins and other antigens.
Current inactivation methods, including the use of formalin, beta-propiolactone, and ultraviolet radiation, have been developed empirically, with little basis in fundamental chemical or structural principles. For example, ethyleneimine monomers have been used to inactivate the foot-and-mouth disease virus (Russian patent no. SU 1915956). Ethyleneimine monomers have also been used to inactivate Mycoplasma and Acholeplasma (WO 92/18161) and avian infections (Romania patent no. RO 101400). Binary ethyleneimine (i.e., ethyleneimine monomer generated by a combination of two reagents) has been used for the inactivation of feline enteric coronavirus, FECV, (EP 94200383). Polyethyleneimine has been used as a plant virus control agent (JP 7882735). The foregoing methods and compounds modify microorganisms, such as viruses and bacteria, nonspecifically, and are difficult to standardize and apply reproducibly.
In addition, ignorance of which chemical alterations render the microorganism noninfectious makes the process difficult to apply reproducibly. Periodic outbreaks of disease resulting from inadequate inactivation or reversion following inactivation are the result. Major outbreaks of paralytic poliomyelitis,, foot and mouth disease and Venezuelan equine encephalitis have occurred due to this problem.
In general, multiple components of the microorganism, including important surface antigenic determinants such as viral capsid proteins, are affected by currently used inactivating agents. These agents significantly modify not only nucleic acids but also other biopolymers such as proteins, carbohydrates and lipids, thereby impairing their function. Altered antigens or the inactivation of protective epitopes can lead to reduced immunogenicity and hence low potency (e.g., inactivated polio vaccine), to altered antigenicity and hence immunopotentiation of disease instead of disease prevention (e.g., respiratory syncytial virus and inactivated measles vaccines produced by formnalin inactivation), or to the appearance of new antigens common to another killed vaccine prepared with the same inactivant.
For example, in the preparation of hepatitis B virus vaccine, it is common practice to heat preparations at temperatures in excess of 80xc2x0 C. and to treat with formaldehyde. These treatments not only inactivate viral infectivity, but also damage proteins and other antigens. Carrier substances added to the vaccine as stabilizers also may be unintentionally modified, producing allergic reactions, as occurs with human serum albumin in rabies vaccine inactivated with beta-propiolactone.
The problems of inactivation of viruses in biological mixtures are distinct from the problems of inactivation of the viruses alone due to the co-presence of desirable biopolymers such as proteins, carbohydrates, and glycoproteins in the plasma. While it is possible to inactivate the hepatitis B virus by using agents such as formaldehyde or oxidizing agents, these methods are not suitable for the inactivation of viruses in blood, due to the observation that most of these inactivating agents impair the biological activity of biopolymers in plasma or cellular components of blood. For example, the use of ultraviolet light has been shown to inactivate viruses in a platelet concentrate. However, severe platelet damage resulted from higher doses. Beta-propiolactone reacts with nucleic acid and protein at similar rates; thus, while viruses can be inactivated, more than half of the factor VIII activity of plasma is lost.
Yet another problem is that some of the viruses contaminating blood or other biological fluids are contained within the cell, either as a fully formed virus, viral genome fragments, or viral nucleic acid integrated into the host genome. For instance, the HIV virus is contained within leukocytes. It is a special concern to be able to inactivate both cell-free and cell-contained forms of virus, while retaining the structural integrity of cells.
Problems may also exist in obtaining valuable biopolymers from non-blood sources since pathogenic viruses may also contaminate such compositions.
The invention features a method of selectively modifying nucleic acid molecules in a biological composition; the method includes the step of contacting the composition with an inactivating agent having the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive. Preferably, R5 is alkylene and each of R1, R2, R3, R4, R6, R7, and R8 is H or alkyl, and n is 2, 3, or 4.
The invention further features a method for selectively inactivating a virus by contacting the biological composition with this inactivating agent, where the nucleic acid molecules are contained within an infectious vertebrate virus. This method may be used for both enveloped and non-enveloped viruses. The inactivated viruses can then be included in killed vaccines.
The invention also features a method for selectively modifying nucleic acids that are contained within a transforming DNA fragment, using this inactivating agent.
The invention also features a killed vaccine that includes an effective amount of inactivated vertebrate virus and a pharmaceutically acceptable carrier, where the inactivated vertebrate virus is made by a process of incubating the virus with an inactivating agent under viral inactivating conditions. The inactivating agent has the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between,1 and 10, inclusive.
The inventions also features a blood-collecting device including a container for receiving blood or a blood fraction; the container includes an inactivating agent in an amount effective to inactivate viruses in the blood or fraction thereof received into the container. The inactivating agent has the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive.
The invention further features a method of selectively modifying nucleic acid molecules in a biological composition; the method includes contacting the composition with an inactivating agent having the formula:
xcfx89xe2x88x92X1xe2x88x92[R1xe2x88x92N+(R2,R3)xe2x88x92]nR4xc2x7(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between,1 and 10, inclusive. Preferably, R1 is alkylene and each of R2, R3, and R4 is H or alkyl.
The invention further features a method for selectively inactivating a virus by contacting the biological composition with this inactivating agent, where the nucleic acid molecules are contained within an infectious vertebrate virus. This method may be used for both enveloped and non-enveloped viruses. The inactivated viruses can then be included in killed vaccines.
The invention also features a method for selectively modifying nucleic acids that are contained within a transforming DNA fragment, using this inactivating agent.
The invention also features a killed vaccine including an effective amount of inactivated vertebrate virus and a pharmaceutically acceptable carrier, where the inactivated vertebrate virus is made by a process of incubating the virus with an inactivating agent under viral inactivating conditions; the inactivating agent has the formula:
xcfx89xe2x88x92X1xe2x88x92[R1xe2x88x92N+(R2, R3)xe2x88x92]nR4xc2x7(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
The invention further features a blood-collecting device including a container for receiving blood or a blood fraction; the container includes an inactivating agent in an amount effective to inactivate viruses in the blood or fraction thereof received into the container. The inactivating agent has the formula:
xcfx89xe2x88x92X1xe2x88x92[R1xe2x88x92N+(R2, R3)xe2x88x92]nR4xc2x7(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive.
The invention also features a nucleic acid inactivating agent having the formula:
xcfx89xe2x88x92X1xe2x88x92[R1xe2x88x92N+(R2, R3)xe2x88x92]nR4xc2x7(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive. Preferably, R1 is alkylene and each of R2, R3, and R4 is H or alkyl.
The invention further features a method of selectively modifying nucleic acid molecules in a biological composition; the method includes the step of contacting the composition with an inactivating agent having the formula:
xcex2xe2x88x92X1xe2x80x94CH2CH2xe2x80x94N30H(R1)xe2x88x92[R2xe2x88x92N+(R3, R4)xe2x88x92]nR519 (X2xe2x88x92)n+1 
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive. Preferably, each of R1, R3, R4, and R5 is H or alkyl and R2 is alkylene.
The invention further features a method for selectively inactivating a virus by contacting the biological composition with this inactivating agent, where the nucleic acid molecules are contained within an infectious vertebrate virus. This method may be used for both enveloped and non-enveloped viruses. The inactivated viruses can then be included in killed vaccines.
The invention also features a method for selectively modifying nucleic acids that are contained within a transforming DNA fragment, using this inactivating agent.
The invention also features a killed vaccine containing an effective amount of inactivated vertebrate virus and a pharmaceutically acceptable carrier, where the inactivated vertebrate virus is made by a process of incubating the virus with an inactivating agent under viral inactivating conditions; the inactivating agent has the formula:
xcex2xe2x88x92X1xe2x80x94CH2CH2xe2x80x94N30H(R1)xe2x88x92[R2xe2x88x92N+(R3R4)xe2x88x92]nR5(X2xe2x88x92)n+1 
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
The invention further features a blood-collecting device that includes a container for receiving blood or a blood fraction; the container includes an inactivating agent in an amount effective to inactivate viruses in the blood or fraction thereof received into the container. The inactivating agent has the formula:
xcex2xe2x88x92X1xe2x80x94CH2CH2xe2x80x94N30H(R1)xe2x88x92[R2xe2x88x92N+(R3R4)xe2x88x92]nR5(X2xe2x88x92)n+1 
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10,inclusive.
Finally, the invention features a nucleic acid inactivating agent having the formula:
xcex2xe2x88x92X1xe2x80x94CH2CH2xe2x80x94N30H(R1)xe2x88x92[R2xe2x88x92N+(R3R4)xe2x88x92]nR5(X2xe2x88x92)n+1 
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. Preferably, each of R1, R3, R4, and R5 is H or alkyl and R2 is alkylene.
xe2x80x9cINACTINE(trademark)xe2x80x9d refers to compounds of the invention having (1) an aziridino moiety or a halo-hydrocarbon-amine moiety, and (2) two or more nitrogen atoms separated by hydrocarbon moieties. These compounds are also referred to as xe2x80x9cinactivating agents,xe2x80x9d or xe2x80x9cselective inactivating agents.xe2x80x9d
An inactivating agent has xe2x80x9cselectivityxe2x80x9d for nucleic acids or xe2x80x9cselectivelyxe2x80x9d reacts with nucleic acids if the comparative rate of reaction of the inactivating agent with nucleic acids is greater than the rate of reaction with other biological molecules, e.g., proteins, carbohydrates or lipids.
xe2x80x9cNucleic acidxe2x80x9d refers to both single and double stranded DNA and RNA.
xe2x80x9cBiological compositionxe2x80x9d refers to a composition containing or derived from cells or biopolymers. Cell-containing compositions include, for example, mammalian blood, red cell concentrates, platelet concentrates, leukocyte concentrates, blood cell proteins, blood plasma, platelet-rich plasma, a plasma concentrate, a precipitate from any fractionation of the plasma, a supernatant from any fractionation of the plasma, blood plasma protein fractions, purified or partially purified blood proteins or other components, serum, semen, mammalian colostrum, milk, saliva, placental extracts, a cryoprecipitate, a cryosupernatant, a cell lysate, mammalian cell culture or culture medium, products of fermentation, ascitic fluid, proteins induced in blood cells, and products produced in cell culture by normal or transformed cells (e.g., via recombinant DNA or monoclonal antibody technology). Biological compositions can be cell-free.
xe2x80x9cBiopolymerxe2x80x9d or xe2x80x9cbiological moleculexe2x80x9d refers to any class of organic molecule normally found in living organisms including, for example, nucleic acids, polypeptides, post-translationally modified proteins (e.g., glycoproteins), polysaccharides, and lipids. Biopolymer-containing compositions include, for example, blood cell proteins, blood plasma, a blood plasma fractionation precipitate, a blood plasma fractionation supernatant, cryoprecipitate, cryosupernatant or portion or derivative thereof, serum, or a non-blood product produced from normal or transformed cells (e.g., via recombinant DNA technology).
xe2x80x9cInhibit the activity of a biopolymerxe2x80x9d means to measurably decrease the function or activity of the biopolymer. The decrease in function or activity can be determined by any standard assay used to measure the activity of the particular biopolymer. For example, the inhibition of an enzyme (protein) or antigen activity can be determined by measuring changes in the rate of an enzymatic process or an immune response to the antigen using conventional assays. Another example of such inhibition is the inhibition of the genome replication, transcription, or translation of an RNA molecule that can be determined by measuring the amount of protein encoded by the RNA that is produced in a suitable in vitro or in vivo translation system.
xe2x80x9cInactivating,xe2x80x9d xe2x80x9cinactivation,xe2x80x9d or xe2x80x9cinactivate,xe2x80x9d when referring to nucleic acids, means to substantially eliminate the template activity of DNA or RNA, for example, by destroying the ability to replicate, transcribe or translate a message. For example, the inhibition of translation of an RNA molecule can be determined by measuring the amount of protein encoded by a definitive amount of RNA produced in a suitable in vitro or in vivo translation system. When referring to viruses, the term means diminishing or eliminating the number of infectious viral particles measured as a decrease in the infectious titer or number of infectious virus particles per ml. Such a decrease in infectious virus particles is determined by assays well known to a person of ordinary skill in the art.
xe2x80x9cViral inactivating conditionsxe2x80x9d refer to the conditions under which the viral particles are incubated with the selective inactivating agents of this invention, including, for example, time of treatment, pH, temperature, salt composition, and concentration of selective inactivating agent, so as to inactivate the viral genome to the desired extent. Viral inactivating conditions are selected from the conditions described below for the selective inactivation of viruses in biological compositions.
xe2x80x9cDiminish infectivity by at least 20 logs by calculationxe2x80x9d means that the decrease in the number of infectious particles is determined by calculation as described herein in Examples 5 and 6.
xe2x80x9cVirusxe2x80x9d refers to DNA and RNA viruses, viroids, and prions. Viruses include both enveloped and non-enveloped viruses, for example, poxviruses. herpes viruses, adenoviruses, papovaviruses, parvoviruses, reoviruses, orbiviruses, picomaviruses, rotaviruses, alphaviruses, rubivirues, influenza virus, type A and B, flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses. rhabdoviruses, lyssaviruses, orthmyxoviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhinoviruses and the filoviruses.
xe2x80x9cVaccinexe2x80x9d is used in its ordinary sense to refer to an agent that is effective to confer the necessary degree of immunity on an organism while causing no morbidity or mortality. Methods of making vaccines are, of course, useful in the study of the immune system and in preventing animal or human disease.
xe2x80x9cPharmaceutically acceptablexe2x80x9d means relatively non-toxic to the animal to whom the compound is administered. xe2x80x9cPharmaceutically acceptable carrierxe2x80x9d encompasses any of the standard pharmaceutical carriers, buffers and excipients such as water, and emulsions, such as oil/water or water/oil emulsions, and various types of wetting agents and/or adjuvants.
The methods and compositions of the present inventions provide advantages over other approaches to selectively modifying nucleic acids in the presence of other biomolecules, and in the presence of cells. As the inactivating agents described herein are selective for the nucleic acids that make up viruses, viruses can be selectively inactivated over the other molecules present.
Other features and advantages of the invention will be apparent from the following description and from the claims.